Structure-based analysis of the β8 interactive sequence of human αB crystallin

The functional importance of the beta 8 sequence (131LTITSSLS138), which is on the surface of the alpha crystallin core domain of human alpha B crystallin, was evaluated using site-directed mutagenesis. Ultraviolet circular dichroism determined that mutating the surface-exposed, nonconserved residues, Leu-131, Thr-132, Thr-134, Ser-135, Ser-136, and Ser-138 individually or in combination (alpha A beta 8 and CE beta 8), had no measurable effect on secondary and tertiary structure. Size exclusion chromatography determined the size of the complexes formed by the beta 8 mutants to be 6-8 subunits larger than wt alpha B crystallin. In chaperone assays, the protective effect of the L131S, T132A, and S135C mutants of the beta 8 sequence was similar to wt alpha B crystallin when, L crystallin and alcohol dehydrogenase were the chaperone substrates and decreased to 66% when citrate synthase was the chaperone substrate. In contrast, the chaperone activity for all three substrates was dramatically reduced for the T134K, S138A, S136H, and CE beta 8 mutants. The prominent location of Thr-134, Ser-136, and Ser-138 on the exposed surface of the alpha crystallin core domain could account for the effect on complex assembly and chaperone activity. Modulation of chaperone activity by the exposed residues of the beta 8 sequence in the alpha crystallin core domain was independent of complex size. The results established the beta 3-beta 8-beta 9 surface of the alpha crystallin core domain as an interface for complex assembly and chaperone activity.